Mycobacterium tuberculosis and Legionella pneumophila ... · been reported. However, the...

INFECTION AND IMMUNITY,0019-9567/00/$04.0010

Sept. 2000, p. 5154–5166 Vol. 68, No. 9

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Mycobacterium tuberculosis and Legionella pneumophila PhagosomesExhibit Arrested Maturation despite Acquisition of Rab7

DANIEL L. CLEMENS,* BAI-YU LEE, AND MARCUS A. HORWITZ

Division of Infectious Diseases, Department of Medicine, UCLA School of Medicine, Center forHealth Sciences, Los Angeles, California 90095

Received 17 March 2000/Returned for modification 5 May 2000/Accepted 19 June 2000

Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocyticpathway. Phagosomes containing inert particles have also been shown to transiently acquire Rab7 as theymature. Disruption in the pathway prior to the acquisition of Rab7 has been suggested as playing a role in thealtered maturation of Mycobacterium bovis BCG phagosomes. As a first step to determine whether disruptionin the delivery or function of Rab7 could play a role in the altered maturation of Legionella pneumophila andM. tuberculosis phagosomes, we have examined the distribution of wild-type Rab7 and the GTPase-deficient,constitutively active mutant form of Rab7 in HeLa cells infected with L. pneumophila or M. tuberculosis. We havefound that the majority of L. pneumophila and M. tuberculosis phagosomes acquire relatively abundant stainingfor Rab7 and for the constitutively active mutant Rab7 in HeLa cells that overexpress these proteins. Never-theless, despite acquisition of wild-type or constitutively active Rab7, both the L. pneumophila and the M.tuberculosis phagosomes continue to exhibit altered maturation as manifested by a failure to acquire lysosome-associated membrane glycoprotein 1. These results demonstrate that L. pneumophila and M. tuberculosisphagosomes have receptors for Rab7 and that the altered maturation of these phagosomes is not due to afailure to acquire Rab7.

Following phagocytosis, phagosomes containing inert parti-cles undergo a series of maturational steps that mirror thestages of the endocytic pathway (16, 17, 36, 37). Immediatelyfollowing phagocytosis, the early phagosomes acquire markersof early endosomes, such as mannose receptor and Rab5 (17,36, 37). With maturation, the phagosomes lose these earlyendocytic markers and acquire markers of late endosomes,such as mannose-6-phosphate receptor and Rab7 (17, 36, 37).With further maturation, the phagosomes lose Rab7 but ac-quire other, as yet unidentified, GTPases (17, 30), fuse withlysosomes, and acquire increasing amounts of lysosomal markerssuch as lysosome-associated membrane glycoproteins (LAMP-1,LAMP-2, and CD63) and acid hydrolases (such as cathepsin Dand acid phosphatase) (17, 36, 37). In addition, phagosomescontaining inert particles acquire the vacuolar proton pumpand, with maturation, become highly acidified (20).

The pathways of phagosomes containing the intracellularparasites Legionella pneumophila and Mycobacterium tubercu-losis deviate markedly from the pathway followed by phago-somes containing inert particles (3, 10, 11, 14, 25, 26, 28, 47).Both L. pneumophila and M. tuberculosis phagosomes resistacidification (14, 28), inhibit phagosome-lysosome fusion (3,26), and acquire little or none of the markers of lysosomes (10,11, 26). Despite these similarities, the L. pneumophila and M.tuberculosis phagosomes also exhibit important differencesfrom one another. The L. pneumophila phagosome, but not theM. tuberculosis phagosome, exhibits unique interactions withother host cell organelles (25). Within minutes of phagocytosis,smooth vesicles appear to be fusing with or budding off of thenascent L. pneumophila phagosome. Subsequently, the L.pneumophila phagosome develops interactions with mitochon-dria, ribosomes, and endoplasmic reticulum (ER), ultimately

forming a ribosome-lined replicative vacuole (25). Whereasthe L. pneumophila phagosome excludes markers of early en-dosomes (11, 13), the M. tuberculosis phagosome shows a per-sistent staining for the transferrin receptor (11) and Rab5 (13)and demonstrates a persistent capacity to acquire exogenouslyadded transferrin from early endosomes (12). A related myco-bacterium, Mycobacterium bovis BCG, has also been shown toexclude LAMP-1 from its phagosome and to demonstrate apersistence of the transferrin receptor and Rab5 on its phago-some (45). Thus, in different ways, both L. pneumophila and M.tuberculosis alter the maturation of their phagosomes and pro-duce a phagosomal environment that is more hospitable totheir growth and multiplication. However, the mechanisms bywhich they do so remain to be elucidated.

Rab-GTPases are members of the Ras superfamily that havebeen shown to play a pivotal role in regulating docking andfusion events between different compartments of eukaryoticcells (23, 34, 35). Because Rab-GTPases play a crucial role inthe regulation of membrane trafficking within eukaryotic cells,disruption of their distribution or function by an intracellularparasite could also play an important role in altering the mat-urational pathway of the phagosome containing the intracellu-lar parasite.

Over 30 different Rab-GTPases have been identified thusfar, and it is thought that every compartment of the secretoryand endocytic pathway has a unique set of Rab-GTPases. Forexample, Rab5 is present on early endosomes (9) and onphagosomes immediately after phagocytosis (16, 17, 30) andregulates membrane trafficking events involving these com-partments (2, 5, 7, 42). Rab7 has been shown to overlap withmannose-6-phosphate-receptor-positive late-endocytic com-partments (9), and the constitutively active Rab7 also shows apartial colocalization with lysosomal compartments (33).Green fluorescence protein-tagged canine Rab7 overexpressedin HeLa cells has also been observed to overlap with lysosomalcompartments (8). The functional importance of Rab7 in reg-ulating membrane trafficking in the endocytic pathway has

* Corresponding author. Mailing address: Division of InfectiousDiseases, Department of Medicine, UCLA School of Medicine, CHS37-121, Los Angeles, CA 90095. Phone: (310) 825-9324. Fax: (310)794-7156. E-mail: [email protected].

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been established by the demonstration that expression of adominant-negative Rab7 mutant interrupts the normal endo-cytic flow from early to late endosomes and causes an accu-mulation of endocytosed vesicular stomatitis virus G protein(18) and cathepsin D and mannose-6-phosphate receptor (38)in early endocytic compartments. In addition, overexpressionof the green fluorescence protein-tagged Rab7 dominant-neg-ative mutant has been shown to lead to dispersal of the lyso-somal compartment and impairment in the capacity of thelysosomes to acidify and to acquire endocytosed material (8).

The role of Rab7 in the formation of L. pneumophila or M.tuberculosis phagosomes in human cells has not previouslybeen reported. However, the acquisition of various Rab-GTPases by L. pneumophila phagosomes (39) or M. bovis BCGphagosomes (45) has been studied in mouse bone marrowmacrophages. In the case of L. pneumophila, Roy et al. (39)recently employed immunofluorescence microscopy to exam-ine the acquisition of Rab7 and LAMP-1 by wild-type and dotAmutant L. pneumophila phagosomes at early times after phago-cytosis. These authors observed that approximately 35% ofwild-type L. pneumophila acquired Rab7 by 5 min after phago-cytosis, and this percentage of L. pneumophila phagosomesbearing Rab7 declined to approximately 10% by 30 min afterphagocytosis. A similar percentage (approximately 45%) of thedotA mutant L. pneumophila phagosomes were observed toacquire Rab7 by 5 min, but essentially all of the dotA mutant L.pneumophila phagosomes lost the Rab7 staining by 30 min inthese experiments. In the case of M. bovis BCG, an avirulentform of a mycobacterial species related to M. tuberculosis, Viaet al. (45) used biochemical techniques to examine Rab-GTPaseson a population of phagosomes isolated from mouse bonemarrow macrophages infected with M. bovis BCG. These in-vestigators reported that M. bovis BCG phagosomes acquireRab5 but not Rab7 or LAMP-1.

To further investigate whether M. tuberculosis or L. pneu-mophila disrupts the maturation of their phagosome by alter-ing the function or distribution of Rab-GTPases, we have ex-amined the distribution of Rab7 in human HeLa cells infectedwith L. pneumophila or M. tuberculosis. We have employedimmunoelectron microscopy, a technique that provides consid-erably more ultrastructural detail than immunofluorescenceand allows one to distinguish whether a phagosome is trulydecorated with a marker or simply surrounded with vesiclesthat bear the marker. The technique allows a determination ofwhether or not individual phagosomes that stain for Rab7 door do not also stain for other markers, such as LAMP-1, aquestion that is not easily addressed with studies of popula-tions of isolated phagosomes. In addition to studying the wild-type Rab7, we have also examined the distribution of a con-stitutively active mutant of Rab7 and the effect of this mutanton the phenotype of the L. pneumophila and M. tuberculosisphagosomes.

MATERIALS AND METHODS

Reagents and antibodies. Glutaraldehyde was purchased from Polysciences(Warrington, Pa.); piperazine-N,N9-bis(2-ethanesulfonic acid) (PIPES), methyl-cellulose, polyvinylpyrrolidone, paraformaldehyde were purchased from SigmaChemical Company (St. Louis, Mo.); Dulbecco’s phosphate-buffered saline waspurchased from Gibco Laboratories (Santa Clara, Calif.); and DMEM (Dulbec-co’s modified Eagle’s Medium) was purchased from Irvine Scientific Co. (SantaAna, Calif.).

Mouse monoclonal antibody (MAb) to the human transferrin receptor (im-munoglobulin G1 [IgG1]) was purchased from AMAC (Westbrook, Maine).Mouse MAb to LAMP-1 (H4A3, IgG1) was obtained from the Hybridoma Bankof the University of Iowa, Iowa City. Mouse MAb to the bovine cation-depen-dent mannose-6-phosphate receptor was provided by Stuart Kornfeld (Washing-ton University, St. Louis, Mo.). Isotypic mouse myeloma control proteins wereobtained from Cappel Organon-Technica (Westchester, Pa.). Rabbit antibody to

mycobacterial lipoarabinonmannan (LAM) was prepared as described previously(11). Rabbit antibody to L. pneumophila lipopolysaccharide (LPS) was preparedby immunizing rabbits with LPS purified from L. pneumophila Philadelphia 1 inFreund’s adjuvant (19). Purified rabbit anti-mouse IgG antibody was obtainedfrom Sigma Chemical Company. Reactivity of this commercial antibody to my-cobacterial antigens was eliminated by three consecutive overnight adsorptionsto an excess of heat-killed M. tuberculosis. Protein A colloidal gold conjugates (5,10, and 15 nm) were provided by G. Posthuma (Utrecht University, The Neth-erlands).

Bacteria. M. tuberculosis Erdman (ATCC 35801), a highly virulent strain, wasobtained from the American Type Culture Collection (Rockville, Md.). Theorganism was passaged through guinea pig lung to maintain virulence, andinfecting inocula were prepared as described previously (13). The concentrationof organisms was determined by measurement of optical density at 540 nm andby counting in a Petroff-Hauser chamber. Viability of the organisms was deter-mined by plating serial dilutions of the infecting inoculum on 7H11 agar. Via-bility ranged from 67 to 86% in these experiments.

L. pneumophila Philadelphia 1 was grown in embryonated hens’ eggs, har-vested, tested for viability and contaminants, and stored at 270°C, as described(29). The egg yolk-grown L. pneumophila was cultured one time only on charcoalyeast extract (CYE) agar, harvested after four days of growth, and used imme-diately. The avirulent mutant L. pneumophila 25D was prepared and maintainedas described previously (27). This mutant has been shown to bear a mutation inthe dot-icm virulence locus (32, 40).

Cloning, expression, and purification of recombinant Rab7 and preparation ofantisera. To clone the human rab7 gene, we screened a human fetal lung cDNAlibrary (Invitrogen) by colony hybridization by using a cDNA probe encoding the39 one-third of a human rab7-like gene on a 240-bp EcoRI-PstI fragment excisedfrom IMAGE Consortium Clone 108659 (ATCC 330444). The probe was labeledwith [a-32P]dCTP (Amersham) by the random priming method. Prehybridizationand hybridization were carried out at 42°C in a solution containing 23 PIPES,50% deionized formamide, 0.5% (wt/vol) sodium dodecyl sulfate (SDS), and 100mg of denatured, sonicated salmon sperm DNA per ml. Positive clones wereselected after three rounds of colony hybridization and were analyzed by restric-tion enzyme digestions. The identities of the positive clones were confirmed bysequencing both strands of DNA in opposite directions. The nucleotide se-quences of our clones were identical at the nucleotide level to the sequence of arab7 gene isolated from a human placenta cDNA library (46), and a similarsequence has also been found by PCR amplification of total mRNA of humanU937 cells (15). The human rab7 gene is highly homologous to the canine rab7sequence (9). The cDNA for the complete rab7 gene was amplified by PCR andcloned into pET3a between NcoI and BamHI cleavage sites. The construct wasunder the control of the T7 promoter with an amino-terminal sequence codingfor a His6 tag. High-level expression of Rab7 in Escherichia coli BL21(DE3) wasinduced with 0.4 mMisopropyl-b-D-thiogalactopyranoside (IPTG), and the re-combinant proteins were purified to homogeneity from sonicated cell pelletextracts by a combination of nickel-affinity, gel filtration, and ion-exchange chro-matography. The resulting material was found by SDS-polyacrylamide gel elec-trophoresis to exhibit a single band of 25 kDa by Coomassie blue staining. Rabbitpolyclonal antibodies to recombinant human Rab7 were raised by immunizingNew Zealand White rabbits three times, 3 weeks apart, with 1 mg of recombinantprotein (purified from E. coli) in Syntex adjuvant (1). This adjuvant was used toavoid the production of antibodies to mycobacterial antigens present in Freund’sadjuvant. The first immunization was supplemented with 100 mg of N-acetylmu-ramyl-L-alanyl-D-isoglutamine (Sigma Chemical Co.). Rabbits immunized withthe recombinant proteins yielded high-titer specific polyclonal antisera to humanRab7. The resulting polyclonal antibodies were affinity purified by binding torecombinant E. coli Rab7, eluted with glycine-HCl, pH 2.5, containing 0.1%bovine serum albumin carrier protein, and immediately neutralized with Tris-HCl, pH 8.0. The purified antibodies reacted equally well with geranylated andnongeranylated Rab7 and did not cross-react with L. pneumophila or M. tuber-culosis antigens. Antisera to Rab7 did not cross-react with Rab5 or Rab4.

Stable transfection of human cell lines with Rab7 and a constitutively activeRab7 mutant. To facilitate the immunolocalization of Rab7 in infected cells, weused the “Tet-off” tetracycline-suppressible expression system (21, 33) to developa stably transfected human HeLa cell line with regulated expression of recom-binant human Rab7 (HeLa-Rab7). We cloned the human rab7 gene into pTRE,transfected the recombinant plasmids into HeLa–Tet-off cells (Clontech) bycalcium phosphate precipitation, and selected stably transfected clones withhygromycin (200 mg/ml) in the presence of tetracycline (5 mg/ml).

GTPase-deficient, fusion-promoting mutant forms of Rab7 and other Rab-GTPases have been described (7, 31). We prepared the corresponding rab7Q67L mutant with PCR-based mutagenesis by published methods (30, 41) byusing the mutant primer 59-GGAACCGTTCAAGTCCTGCTGTGTCCCATAT-39 (mutated nucleotides underlined) and pTRE forward and reverse sequences(59-TCCAGCCTCCGCGGCCCC-39) and 59-TCATCAATGTATCTTATCATGTCT-39, respectively) as outer primers in the amplifications. The mutant con-struct was confirmed by DNA sequencing. HeLa cells stably transfected with theconstitutively active rab7 (HeLa-Rab7 Q67L) were selected as described abovefor the wild-type rab7.

Transient transfection of HeLa cells with the bovine cation-dependent man-nose-6-phosphate receptor. A 1.1-kb EcoRI-PstI fragment containing the bovine

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cation-dependent mannose-6-phosphate receptor (CD-M6PR) was releasedfrom pSV-neo-cdmpr (provided by Stuart Kornfeld, Washington University) andsubcloned into pcDNA3.1/Zeo(1) (Invitrogen). Coexpression of Rab7 and CD-M6PR was obtained by transfecting HeLa-Rab7 or HeLa-Rab7 Q67L cells withpcDNA3.1/Zeo(1)-cdmpr. Transfected cells were kept in the absence of antibi-otics for 2 days before infection with M. tuberculosis and L. pneumophila. Im-munofluorescence microscopy demonstrated partial colocalization of the Rab7and the CD-M6PR, consistent with published observations of other investigators(9).

Assessment of intracellular growth of M. tuberculosis and L. pneumophila inmonolayers of THP-1 cells or HeLa cells. Monolayers of HeLa cells (105 cells/well) or THP-1 cells (4 3 105 cells/well) were cultured to confluency in 2-cm2

tissue culture wells for 2 days in RPMI 1640 (THP-1) or DMEM (HeLa) with10% fetal bovine serum without tetracycline. The THP-1 cells were differentiatedwith phorbol myristic acid (0.16 nM). Monolayers were coincubated with eitherM. tuberculosis (106/ml) or L. pneumophila (2 3 107/ml or 2 3 108/ml). Mono-layers were washed with culture medium and incubated in fresh medium at 37°C.CFU of M. tuberculosis were determined at sequential time points using themethod described by Hirsch et al. (24). Briefly, the supernatant fluid was re-moved and retained, and the monocyte monolayer was lysed by adding 0.1% SDSin sterile distilled water. The tissue culture supernatant fluid and the lysate of thecell monolayer were combined, and serial dilutions were plated on 7H11 agar for2 weeks at 37°C in 5% CO2, and CFU were enumerated. In the case of L.pneumophila, CFU were determined by a modification of the method of Horwitzand Silverstein (29). Briefly, culture media in the wells were removed, saved, andreplaced with sterile distilled water, and the monolayers of infected cells werelysed with a 2-mm probe tip sonicator (Heat Systems-Ultrasonics, Plainview,N.Y.) at setting 4 for 10 s. This amount of sonic energy lysed the HeLa cellscompletely, as determined by phase-contrast microscopy, but did not reduce L.pneumophila CFU. The culture supernate and the hypotonic sonicate were com-bined, serially diluted, and plated on CYE agar plates.

Infection of monolayers of HeLa cells with M. tuberculosis and L. pneumophila.Stably transfected HeLa cells were plated at a density of 2.5 3 106 per 75-cm2

culture flask or at a density of 106 per 10-cm-diameter tissue culture plate.Optimal Rab7 expression was obtained by omitting tetracycline from the culturemedium 1 to 3 days before the cells were to be fixed. Monolayers were culturedwithout antibiotics in DMEM (low glucose) with 10% fetal calf serum (certifiedtetracycline negative) (Clontech). To examine the interaction of HeLa cells withL. pneumophila, we plated stably transfected HeLa cells in 10-cm-diameter petriplates in DMEM containing 10% heat-inactivated fetal bovine serum withouttetracycline. Two days later, the plates were chilled on ice, and suspensions ofwild-type or avirulent L. pneumophila (2 3 109/ml) were added to the plates at0°C. The plates were centrifuged for 20 min at 1,160 3 g in a biohazard-safe

rotor, were incubated at 37°C for 30 min (L. pneumophila), and were either fixedimmediately or washed extensively and incubated for an additional 15 min to 8 hprior to fixation. To examine the interaction of HeLa cells with M. tuberculosis,we coincubated the HeLa cells with live or heat-killed M. tuberculosis (4 3108/ml) for 2 h at 37°C. Subsequently, the monolayers were washed extensivelywith culture medium to remove noningested bacteria and beads, the medium wasreplaced with fresh DMEM containing 10% fetal calf serum, and the monolayerswere incubated for 1 to 3 days prior to fixation. In some experiments, 1-mm-diameter latex beads (1:500 dilution of a 2.5% solid suspension) were addedduring coincubation with L. pneumophila or M. tuberculosis.

Immunoelectron microscopy. Monolayers were fixed with 2% paraformalde-hyde in 0.1 M PIPES, pH 7.3, containing 6% sucrose for 2 h at 4°C and processedfor cryoimmunoelectron microscopy as described previously (13). Cryosectionswere collected on formvar-coated nickel grids, blocked with 1% bovine serumalbumin, 0.1% fish skin gelatin in 0.05 M HEPES, pH 7.5, containing 0.3 M NaClfor 1 h at 4°C. Immunogold double and triple labelings were performed by amodification of the sequential protein A gold technique as described by Slot etal. (41). Briefly, sections were stained first for Rab7 by using 15-nm-diameterprotein A gold particles and were washed, and free protein A sites were de-stroyed by incubation with 2% glutaraldehyde for 10 min. Aldehydes werequenched by floating grids face down for 5 min on each of two consecutive dropsof 50 mM glycine in phosphate-buffered saline. The sections were then incubatedfirst with rabbit anti-LAM or anti-LPS and then with 5-nm-diameter protein Agold particles. Staining for the cation-dependent mannose-6-phosphate receptor,LAMP-1, or LAMP-2 was accomplished by incubating sections with primarymouse MAb (10 mg/ml) in blocking buffer for 1 h (22°C), washing, and incubatingwith rabbit anti-mouse IgG, (45 min at 22°C) followed by incubation with 10-nm-diameter protein A gold particles (30 min at 22°C). Sections were embeddedin 1.8% methylcellulose–0.4% uranyl acetate (22). Consecutive phagosomeswere photomicrographed with a JEOL 100 CX II electron microscope. Mea-surements of gold particles per micrometer of membrane or per square micro-meter of cytoplasm were made with a Numonics 2210 digitizer tablet and Sig-maScan software (Jandel Scientific Co., Corte Madera, Calif.).

RESULTS

Establishment of a model human cell system suitable forevaluating Rab7 expression on phagosomes. We have foundthat endogenous levels of Rab5 and Rab7 in normal humanmonocytes, monocyte-derived macrophages, and cell lines aretoo low to be detected reliably by immunofluorescence or

FIG. 1. Growth of L. pneumophila and M. tuberculosis in HeLa–Tet-off cells or in Hela-Rab7 or HeLa-Rab7 Q67L cells overexpressing Rab7 or Rab7 Q67L.Monolayers of HeLa–Tet-off, HeLa-Rab7, and HeLa-Rab7 Q67L cells in 2-cm2 wells were grown for 2 days (A) or 1 day (B) in the absence of tetracycline to induceexpression of Rab7 or Rab7 Q67L. The cells were incubated with L. pneumophila (2 3 107/ml [A]) or M. tuberculosis (106/ml [B]) for 2 h at 37°C, were washed, andwere incubated in fresh medium at 37°C. At sequential times thereafter, the monolayers were lysed and combined with the culture supernatant, and CFU weredetermined by plating serial dilutions on CYE (A) and 7H11 (B) agar plates. Data shown represent the means 6 the standard deviations of triplicate determinations.

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immunoelectron microscopy. Therefore, to study the distribu-tion and function of these Rab-GTPases in host cells infectedwith intracellular pathogens, we cloned the rab5 (13) and rab7genes from a human fetal lung library and sought to overex-press the genes in a variety of different cell types. Since mac-rophages are the natural host cells of L. pneumophila and M.tuberculosis, we initially sought to overexpress the rab genes inmacrophage-like cell lines (U937, THP-1, and HL60) but wereunable to achieve stable high-level expression compatible withimmunofluorescence or immunoelectron microscopy studies.We therefore prepared a HeLa cell line capable of inducible

expression of the human rab5 (13) and rab7 genes. Becauselong-term overexpression of Rab-GTPases is often associatedwith toxicity and loss of expression by the cell line, we used atetracycline-regulated expression system (21). Expression ofRab7 and the constitutively active Q67L form of Rab7 by theisolated clones was tightly regulated by tetracycline and wasstable for at least 3 days after removal of tetracycline from theculture medium (data not shown).

To confirm that the study of L. pneumophila and M. tuber-culosis phagosomes in infected HeLa cells is relevant to un-derstanding the pathogenesis of L. pneumophila and M. tuber-

FIG. 2. Quantitation of Rab7 and LAMP-1 immunogold staining in HeLa-Rab7 cells infected with wild-type or avirulent L. pneumophila. Wild-type or avirulentL. pneumophila cells were spun down onto monolayers of HeLa-Rab7 cells at 4°C. The monolayers were incubated for 30 min at 37°C and were either fixed immediately(0 min) or washed to remove nonadherent bacteria and were incubated at 37°C for an additional 15 min, 1 h, 3 h, or 8 h, as indicated in the figure, and were then fixed.All monolayers were processed for cryoimmunoelectron microscopy. Rab7 (A) and LAMP-1 (B) immunogold particles were enumerated on phagosomal membranes,nuclear membranes, and plasma membranes, and the total number of immunogold particles per square micrometer of cytoplasm (including all cytoplasmic vesicles,phagosomes, and other organelles) was also determined. Data shown represent the means and standard errors of the means of gold particle counts for eachcompartment. (A, left panel) Rab7 was present on wild-type L. pneumophila phagosomes for the first 3 h after infection, but was scarce at 8 h after infection. Rab7was also present on avirulent L. pneumophila phagosomes, but at somewhat lower overall levels than found on the wild-type L. pneumophila phagosomes. AvirulentL. pneumophila phagosomes were not examined at the 8-h time point. Rab7 was scarce on nuclear membranes and plasma membranes at all time points examined. Rightpanel, as a control, Rab7 staining in the cytoplasm of the HeLa cells was quantitated and found to be comparable in the cells containing wild-type or avirulent L.pneumophila. (B, left panel) LAMP-1 was scarce on wild-type L. pneumophila phagosomes at all time points examined but was abundant on phagosomes containingthe avirulent L. pneumophila. Plasma membranes and nuclear membranes served as internal negative controls and had negligible staining for LAMP-1. Right panel,LAMP-1 staining per unit area of cytoplasm was similar between HeLa cells infected with wild-type and avirulent L. pneumophila. Total numbers of phagosomesevaluated were as follows: 29, 72, 44, 63, and 42 for wild-type L. pneumophila at 0 min, 15 min, 1 h, 3 h, and 8 h, respectively, and 29, 54, and 63 for avirulent L.pneumophila at 0, 1, and 3 h, respectively.



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culosis infection of macrophages, the natural host cells of thesepathogens in humans, we investigated the extent to which theinteraction of the pathogens with HeLa cells resembles theirinteraction with human macrophages (13). We examined thecapacity of the two pathogens to multiply within HeLa–Tet-offcells and HeLa–Tet-off cells expressing Rab5 (HeLa-Rab5cells) and the expression of endolysosomal markers on thephagosomes of the two pathogens in the HeLa cells.

(i) Intracellular growth of L. pneumophila and M. tuberculo-sis in HeLa–Tet-off, HeLa-Rab7, and HeLa-Rab7 Q67L cells.We found that although HeLa cells are poorly phagocytic,adequate uptake of L. pneumophila and M. tuberculosis couldbe obtained by increasing the multiplicity of infection relativeto that used when infecting more phagocytic cells. Once takenup by the HeLa–Tet-off or HeLa-Rab5 cells, L. pneumophilaand M. tuberculosis grow at rates comparable to that in THP-1cells as described (13).

To determine whether overexpression of Rab7 or Rab7Q67L alters the growth of L. pneumophila or M. tuberculosis inHeLa cells, we examined the growth of L. pneumophila and M.tuberculosis in HeLa–Tet-off, HeLa-Rab7, and HeLa-Rab7Q67L cells 1 day after withdrawal of tetracycline (Fig. 1A andB). Overexpression of Rab7 or Rab7 Q67L did not alter thegrowth of L. pneumophila or M. tuberculosis in the HeLa cells.

(ii) Distribution of endocytic markers on L. pneumophila orM. tuberculosis phagosomes in HeLa cells. To determine ifphagosomes in infected HeLa cells have molecular character-istics similar to phagosomes in infected human macrophages,we have previously examined transferrin receptor expressionon M. tuberculosis phagosomes and LAMP-1 expression onboth L. pneumophila and M. tuberculosis phagosomes (13).Consistent with our published observations with human mono-cyte-derived macrophages (11), we found that the majority ofM. tuberculosis phagosomes in HeLa-Rab5 cells stably trans-fected with the transferrin receptor gene stained positive forthe transferrin receptor (13). Also consistent with our previousobservations in human macrophages (11), we found little or noLAMP-1 on phagosomes containing wild-type L. pneumophilaor live M. tuberculosis in HeLa-Rab5 cells, but intense stainingfor LAMP-1 on phagosomes containing the avirulent mutantL. pneumophila, heat-killed M. tuberculosis, or latex beads inthese cells (13). These results confirmed that L. pneumophila

and M. tuberculosis phagosomes in HeLa-Rab5 cells do notfuse with lysosomes and that overexpression of the Rab5 inHeLa cells does not fundamentally alter the membrane-traf-ficking properties of the L. pneumophila or M. tuberculosisphagosomes.

We concluded from these sets of studies that, while uptakeof L. pneumophila and M. tuberculosis into HeLa cells is muchless efficient than uptake into macrophages, the intracellularrates of bacterial growth and the interaction of the phago-somes with the endolysosomal pathway in these host cells arevery similar. This implied that lessons learned from studying L.pneumophila and M. tuberculosis phagosomes in HeLa cellswere likely to apply to phagosomes of these pathogens inmacrophages.

Distribution of Rab7 on phagosomes containing wild-typeand avirulent L. pneumophila in HeLa-Rab7 cells. Two daysafter removal of tetracycline from the culture medium, 90% ofHeLa-Rab7 cells had abundant immunogold staining for Rab7on cytoplasmic vesicles (.1 gold particle/mm2), with an aver-age level of staining of 4.8 6 0.6 gold particles/mm2 (Fig. 2A,right side of panel). In contrast, parental HeLa–Tet-off cellshad only a low level of staining for endogenous Rab7 (averagelevel of cytoplasmic immunogold staining 5 0.21 gold particle/mm2) (data not shown). In HeLa-Rab7 cells infected with wild-type L. pneumophila, the majority of L. pneumophila phago-somes stained positive for Rab7 for at least the first 3 h afterinfection (Fig. 2A and 3A). Although there was heterogeneityin the levels of staining for Rab7 (Fig. 3), during the first 3 h,60% of the phagosomes had more immunogold staining than90% of the nuclei in the same cells. However, by 8 h afterinfection, the Rab7 immunogold staining dropped to very lowlevels, with no significant difference between the low levels ofphagosomal or nuclear staining (Fig. 2). Avirulent L. pneumo-phila phagosomes had lower levels of staining for Rab7 thanthe wild-type phagosomes (Fig. 2A), but a majority of bothtypes of phagosomes stained positive. While wild-type andavirulent phagosomes displayed a similar distribution of stain-ing during the first 3 h after phagocytosis (Fig. 3A and B), noneof the avirulent phagosomes exhibited the strikingly high levelsof Rab7 staining (.3 gold particles/mm) found on 15 to 20% ofthe wild-type phagosomes.

FIG. 3. Distribution of staining for Rab7 and LAMP-1 in HeLa-Rab7 cells fixed 3 h after infection with either wild-type or avirulent L. pneumophila. As describedfor Fig. 2, wild-type (A and C) or avirulent (B and D) L. pneumophila cells were spun down onto monolayers of HeLa-Rab7 cells at 4°C. The monolayers were incubatedfor 30 min at 37°C, were washed extensively, were incubated 2 h at 37°C, were fixed, and were processed for cryoimmunoelectron microcopy. Gold particles permicrometer of membrane on phagosomal membranes, plasma membranes, and nuclear membranes were enumerated for Rab7 (A and B) and LAMP-1 (C and D).



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Distribution of LAMP-1 on phagosomes containing wild-type or avirulent L. pneumophila in HeLa-Rab7 cells. Little orno LAMP-1 immunogold staining was observed on the wild-type L. pneumophila phagosomes at all time points examined,from 0 min to 8 h (Fig. 2B and 3C). Even L. pneumophilaphagosomes that stained positively for Rab7 showed little orno immunogold staining for LAMP-1 (Fig. 4A). In contrast,avirulent L. pneumophila phagosomes showed high levels ofimmunogold staining for LAMP-1 (Fig. 2B, 3D, and 4B).

Distribution of Rab7 on phagosomes containing live andheat-killed M. tuberculosis cells in HeLa-Rab7 cells. In HeLa-Rab7 cells infected with M. tuberculosis, a substantial propor-tion of M. tuberculosis phagosomes stained positive for Rab7 atall time points examined, from 1 to 3 days after phagocytosis(Fig. 5A, 6A, and 7). Although there was heterogeneity in theintensity of staining, 65% of the M. tuberculosis phagosomeshad a higher level of Rab7 immunogold staining than thenuclei within the same cells (Fig. 6A). The majority of phago-somes containing heat-killed M. tuberculosis also stained pos-itive for Rab7, but tended to have a lower level of staining thanthe phagosomes containing live M. tuberculosis (Fig. 5A and6B).

Distribution of LAMP-1 on phagosomes containing live orheat-killed M. tuberculosis in HeLa-Rab7 cells. Despite the

presence of relatively high levels of Rab7 on the phagosomescontaining live M. tuberculosis, the majority of such phago-somes acquired only low levels of LAMP-1 (Fig. 5B, 6C, and7). Phagosomes containing heat-killed M. tuberculosis, on theother hand, acquired abundant staining for LAMP-1 (Fig. 5Band 6D).

It is possible that phagosomes containing heat-killed M. tu-berculosis would have exhibited higher levels of staining forRab7 at earlier time points, when phagosomes containing inertparticles might be expected to have more Rab7. However, wewere unable to examine levels at earlier time points because, atthe multiplicities of infection used, heat-killed M. tuberculosiswas taken up inefficiently by HeLa cells.

Effect of overexpression of the Rab7 constitutively activemutant on L. pneumophila and M. tuberculosis phagosomes.Whereas wild-type Rab7 exhibits only a limited colocalizationwith LAMP-1, the GTPase deficient, constitutively active Rab7Q67L mutant exhibits a much greater degree of colocalizationwith lysosomal compartments (33). Therefore, we examinedthe distribution of the constitutively active Rab7 Q67L in L.pneumophila- and M. tuberculosis-infected cells and the effectof expression of the constitutively active Rab7 on the phago-somal phenotype. We found that the constitutively active Rab7is recruited to L. pneumophila (Fig. 8A and see Fig. 10B and C)and M. tuberculosis phagosomes (Fig. 9A and 10D and E), justas was the case with the wild-type Rab7. At 2 h after infection,the level of Rab7 Q67L present on the avirulent L. pneumo-phila phagosomes was similar to the level found on wild-type L.pneumophila phagosomes (Fig. 8A). Despite similar levels ofRab7 Q67L on wild-type and avirulent L. pneumophila phago-somes, LAMP-1 was scarce on the wild-type L. pneumophilaphagosomes but abundant on the avirulent L. pneumophilaphagosomes (Fig. 8B and 10B and C). As expected, Rab7Q67L colocalized extensively with LAMP-1 in cytoplasmic ves-icles of the cells infected with wild-type L. pneumophila; manyof these vesicles appeared to be autophagosomes or multive-sicular bodies (Fig. 10A). Nevertheless, wild-type L. pneumo-phila phagosomes that stained richly for Rab7 Q67L did notacquire LAMP-1 (10B). Similarly, the levels of Rab7 Q67Lfound on live M. tuberculosis phagosomes were similar to orgreater than the levels found on latex bead phagosomes in thesame cells (Fig. 9). Nevertheless, M. tuberculosis phagosomesthat stained positive for Rab7 Q67L exhibited little or noimmunogold staining for LAMP-1, whereas latex bead phago-somes acquired abundant amounts of LAMP-1 (Fig. 9 and 10Cand D).

While we have found that overexpression of constitutivelyactive Rab5 resulted in dramatic changes in morphology andmembrane markers of M. tuberculosis phagosomes (12), over-expression of the constitutively active form of Rab7 caused nochange in the phenotype of L. pneumophila or M. tuberculosisphagosomes, despite the presence of extremely rich stainingfor the constitutively active Rab7 on the phagosomes (Fig.10B, D, and E).

Distribution of the cation-dependent mannose-6-phosphatereceptor in HeLa cells infected with L. pneumophila or M.tuberculosis. Both Rab7 and the cation-dependent mannose-6-phosphate receptor (CD-M6PR) are present on late endo-somes. To determine if L. pneumophila and M. tuberculosisarrest the maturation of their phagosomes at a CD-M6PR1/LAMP2 late endosomal stage, we examined whether L. pneu-mophila or M. tuberculosis phagosomes that stain intensely forRab7 or Rab7 Q67L would also stain for the CD-M6PR. HeLaRab7 and HeLa Rab7 Q67L cells were transiently transfectedwith the gene for the CD-M6PR and were infected with L.pneumophila or M. tuberculosis. In the case of L. pneumophila

FIG. 4. Phagosomes containing wild-type L. pneumophila, but not avirulentL. pneumophila, lack LAMP-1 despite recruitment of Rab7. Monolayers ofHeLa-Rab7 cells were coincubated with latex beads and wild-type (A) or avir-ulent (B) L. pneumophila, fixed, and processed for immunoelectron microscopy3 h after infection. Rab7 was stained with 15-nm gold particles (large arrow-heads), LAMP-1 was stained with 10-nm gold particles (small arrowheads), andL. pneumophila LPS was stained with 5-nm gold particles (small arrows). (A)Two wild-type L. pneumophila phagosomes and one latex bead phagosome areshown in this micrograph. The wild-type L. pneumophila phagosome on the lefthas four Rab7 immunogold particles, and the phagosome on the right has oneRab7 immunogold particle. Neither L. pneumophila phagosome has anyLAMP-1 immunogold staining. The latex bead phagosome located between thetwo L. pneumophila phagosomes has two Rab7 immunogold particles, and ap-proximately 20 LAMP-1 immunogold particles. (B) Two avirulent L. pneumo-phila phagosomes are shown in this micrograph. The avirulent L. pneumophilaphagosome on the left has five Rab7 immunogold particles, and the phagosomeon the right has one Rab7 immunogold particle. Both avirulent L. pneumophilaphagosomes stain intensely for LAMP-1. Magnifications are (A) 326,220 and(B) 324,681.



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phagosomes within HeLa-Rab7 cells expressing CD-M6PR, wefound no significant colocalization of the CD-M6PR with theL. pneumophila phagosomes, despite intense staining of othercytoplasmic vesicles within the cells for the CD-M6PR (Fig.11A; mean level of phagosomal membrane staining 5 0.08 60.06 CD-M6PR gold particles/mm; mean level of nuclear stain-ing 5 0.13 6 0.04 CD-M6PR gold particles/mm; mean level ofcytoplasmic staining 5 13.1 6 3.6 M6PR gold particles/mm2).In the case of M. tuberculosis phagosomes within HeLa-Rab7cells expressing the CD-M6PR, M. tuberculosis phagosomalmembranes displayed a low level of staining for CD-M6PR(Fig. 11B; 0.88 6 0.18 gold particles/mm, compared with nu-clear membrane staining of 0.13 6 0.11 gold particles/mm; P 50.007, unpaired t test). This low level of staining for the CD-M6PR is comparable to the relatively low level seen forLAMP-1 on these phagosomes and was unimpressive com-pared with the overall level of cytoplasmic staining (10.3 6 1.9

gold particles/mm2) and the abundant staining for CD-M6PRseen on adjacent cytoplasmic vesicles (Fig. 11B). Similarly, inHeLa Rab7-Q67L cells, CD-M6PR was absent from L. pneu-mophila phagosomes and scarce on M. tuberculosis phago-somes (data not shown). We have also examined immunogoldstaining for endogenous CD-M6PR and the cation-indepen-dent M6PR in human monocyte-derived macrophages andfound that staining for these markers is scarce on the M.tuberculosis phagosome (data not shown), in agreement withthe findings of this study and that of Xu et al. (47).

DISCUSSION

L. pneumophila and M. tuberculosis both prevent the matu-ration of their phagosomes to phagolysosomes. Part of thenormal maturation of phagosomes containing inert particles tophagolysosomes involves the acquisition of Rab7 (16, 17, 30).

FIG. 5. Quantitation of Rab7 and LAMP-1 immunogold staining in HeLa-Rab7 cells infected with live or heat-killed M. tuberculosis. Monolayers of HeLa-Rab7cells were incubated with suspensions of live or heat-killed M. tuberculosis for 2 h at 37°C, were washed to remove nonadherent bacteria, were incubated for anadditional 1 to 3 days, were fixed, and were prepared for cryoimmunoelectron microscopy. Rab7 (A) and LAMP-1 (B) immunogold particles were enumerated onphagosomal membranes, nuclear membranes, and plasma membranes. Data shown represent the means and standard errors of the means of gold particle counts foreach compartment. (A, left panel) Rab7 was present on phagosomes containing live M. tuberculosis at 1 to 3 days and was also present on phagosomes containingheat-killed M. tuberculosis, but at a somewhat lower level. Rab7 staining was scarce on nuclear membranes and plasma membranes. (A, right panel) As a control, Rab7staining per unit area of cytoplasm of the HeLa cells was quantitated and found to be comparable in cells containing live or heat-killed M. tuberculosis. The level ofstaining in the cytoplasm increased somewhat from 1 to 3 days due to longer tetracycline-free induction. (B, left panel) Live M. tuberculosis phagosomes had low levelsof staining for LAMP-1. In contrast, heat-killed M. tuberculosis phagosomes stained richly for LAMP-1. Plasma membranes and nuclear membranes lacked LAMP-1staining. (B, right panel) LAMP-1 per unit area of cytoplasm was comparable in HeLa cells containing live or heat-killed M. tuberculosis. The total number ofphagosomes evaluated were as follows: 41, 112, and 61 for live M. tuberculosis at 1, 2, and 3 days, respectively, and 85 and 38 for dead M. tuberculosis at 1 and 2 days,respectively.



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To determine if L. pneumophila or M. tuberculosis phagosomesblock this stage of maturation, we examined whether or nottheir phagosomes had the capacity to acquire Rab7. We foundthat the majority of L. pneumophila and M. tuberculosis phago-somes did acquire staining both for Rab7 and for the consti-tutively active mutant form of Rab7 (Rab7 Q67L) in HeLacells overexpressing these proteins. Moreover, phagosomescontaining wild-type L. pneumophila or live M. tuberculosistended to have more Rab7 than phagosomes containing avir-ulent L. pneumophila or heat-killed M. tuberculosis cells, re-spectively. Despite their relatively abundant acquisition ofRab7 and Rab7 Q67L, phagosomes containing wild-type L.pneumophila and live M. tuberculosis acquired little or no stain-ing for LAMP-1. In contrast, phagosomes containing avirulent

L. pneumophila, heat-killed M. tuberculosis, and latex beadsacquired intense staining for LAMP-1.

Prior to our studies, a plausible hypothesis for the capacityof L. pneumophila and M. tuberculosis to alter the maturationof their phagosomes was that these pathogens either preventedtheir phagosomes from acquiring Rab7 or prevented the Rab7from binding to GTP. However, our results render these hy-potheses untenable. While it is likely that the remarkably highlevels of staining for Rab7 on L. pneumophila and M. tubercu-losis phagosomes are unique to host cells that overexpressRab7, our results demonstrate that (i) the L. pneumophila andM. tuberculosis phagosomes have receptors that allow thephagosomes to recruit Rab7 and (ii) the altered maturation ofthe L. pneumophila and M. tuberculosis phagosomes is not due

FIG. 6. Distribution of staining for Rab7 and LAMP-1 in HeLa-Rab7 cells 2 days after infection with live or heat-killed M. tuberculosis. HeLa-Rab7 cells expressingRab7 were incubated with live (A and C) or heat-killed (B and D) M. tuberculosis for 2 h, were washed extensively, were incubated at 37°C for 2 days, were fixed, andwere processed for cryoimmunoelectron microscopy. Gold particles per micrometer of membrane on phagosomal membranes and nuclear membranes are shown forRab7 (A and B) and for LAMP-1 (C and D).



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to a failure to recruit Rab7 to the phagosomes. Clearly, both L.pneumophila and M. tuberculosis phagosomes exhibit alteredmaturation despite the acquisition of either the wild-type Rab7or the constitutively active mutant (GTP-bound) form of Rab7.The recruitment of Rab7 to the phagosomes in our system isspecific and is not due simply to abundance of the Rab7 in thecytoplasm, since nuclear membranes and plasma membranesin these cells showed only low levels of staining for Rab7.

Our observations regarding M. tuberculosis phagosomes inHeLa cells differ markedly from those of Via et al. (45) withavirulent M. bovis BCG phagosomes in mouse macrophages.Via and coworkers reported that M. bovis BCG phagosomesdid not acquire Rab7 and concluded that maturation of theBCG phagosome was blocked at a stage between the acquisi-tion of Rab5 and Rab7. However, in our system, using virulentM. tuberculosis in human HeLa cells, we found that M. tuber-culosis can arrest the maturation of its phagosome at a stagesubsequent to the acquisition of Rab7. These different obser-vations could reflect different model systems (M. bovis BCG inmouse macrophages versus M. tuberculosis in human HeLacells that overexpress Rab7) or the difference in detectionmethod (subcellular fractionation versus immunoelectron mi-croscopy). Failure to observe Rab7 on the M. bovis BCGphagosome could be due to loss of Rab7 during the lengthyprocedure used to isolate the phagosomes (which includes a15-h ultracentrifugation step) or to the sensitivity of the tech-nique. In our system, the levels of Rab7 on the M. tuberculosisphagosome may be exaggerated by the use of transfected cellsthat overexpress the protein. In addition, cycling of an overex-pressed Rab-GTPase theoretically could be impaired if thechaperon proteins, such as Rab-GDI and Rab escort protein,are stoichiometrically overwhelmed, thereby leading to thepersistence of higher levels of the Rab protein on the phago-some. Nevertheless, recruitment of Rab-GTPases requires spe-cific receptor machinery on the target membrane (4, 44), andthe presence of Rab7 on the M. tuberculosis phagosome cannot

be accounted for by either overexpression of the Rab proteinor impaired cycling from the membrane by chaperon proteins.Thus, our data demonstrate that the M. tuberculosis phago-some has receptor machinery for Rab7 and that M. tuberculosiscan block the maturation of its phagosome at a step subse-quent to Rab7 acquisition.

Our observations on the recruitment of Rab7 to L. pneumo-phila phagosomes in HeLa cells by immunoelectron micros-copy are generally consistent with the findings of Roy et al.(39) who also observed Rab7 immunofluorescence on a sub-stantial percentage of L. pneumophila phagosomes in mousebone marrow macrophages. Although we observed a largerpercentage of L. pneumophila phagosomes to be Rab71, thisprobably reflects differences in model systems and detection

FIG. 7. M. tuberculosis phagosomes in HeLa-Rab7 cells stain positively forRab7 but exclude LAMP-1. HeLa-Rab7 cells were maintained and expanded inthe presence of tetracycline (5 mg/ml). One day prior to infection with M.tuberculosis, tetracycline was removed from the culture medium to induce Rab7expression. The HeLa cells were incubated for 2 h with M. tuberculosis using amultiplicity of infection of 50:1. Nonadherent bacteria were washed away, andthe monolayers were incubated for 2 additional days. Monolayers were fixed andprocessed for cryoimmunoelectron microscopy. Rab7 was stained with 15-nmimmunogold particles (large arrowheads) and is abundant on the M. tuberculosisphagosomal membranes. Mycobacterial lipoarabinomannan was stained with5-nm gold particles and is present on the mycobacterial cell wall (small arrows).LAMP-1 immunogold staining (10-nm gold particles) is scarce on the M. tuber-culosis phagosomal membrane but is present on adjacent cytoplasmic vesicles(small arrowheads). Magnification, 333,930.

FIG. 8. Quantitation of Rab7 Q67L and LAMP-1 immunogold staining inHeLa-Rab7 Q67L cells after infection with wild-type or avirulent L. pneumo-phila. Monolayers of HeLa cells expressing Rab7 Q67L were infected withwild-type or avirulent L. pneumophila as described in the legend of Fig. 2. Cellswere fixed at 2 to 5 h after infection and were processed for immunoelectronmicroscopy. Immunogold staining for Rab7 Q67L (A) and LAMP-1 (B) wasquantitated on phagosomal membranes, plasma membranes, and nuclear mem-branes. (A, left panel) Wild-type L. pneumophila phagosomes stained positive forRab7 Q67L at 2 to 3 h, with an overall level of staining similar to that found onavirulent L. pneumophila phagosomes at 2 h. However, Rab7 Q67L levels onwild-type L. pneumophila phagosomes fell to background levels at 5 h afterinfection. Nuclear membranes and plasma membranes served as negative con-trols and had relatively low levels of staining for Rab7 Q67L. (B, left panel)LAMP-1 staining was excluded from wild-type L. pneumophila phagosomes butwas abundant on avirulent L. pneumophila phagosomes. Plasma membrane andnuclear membrane staining served as internal negative controls. (A and B, rightpanels) Immunogold staining per unit area of cytoplasm was counted as apositive control and found to be comparable under the two infection conditionsboth for Rab7 Q67L (A) and for LAMP-1 (B). Data shown represent the meansand standard errors of the means of gold particle counts for each compartment.Total numbers of phagosomes evaluated were as follows: 32, 19, and 38 forwild-type L. pneumophila at 2, 3, and 5 h, respectively, and 45 for avirulent L.pneumophila at 2 h.



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techniques. In both our study and that of Roy et al., wild-typeL. pneumophila phagosomes displayed a trend to lose Rab7immunostaining with time, suggesting that the wild-type L.pneumophila phagosome loses receptors for the Rab7-GTPaseby 5 to 8 h after infection, a time period corresponding to thatin which L. pneumophila completes the formation of its ribo-some-lined replicative vacuole (25). Our studies have ex-panded upon the observations made by Roy et al. (39) byexamining the phenotype of L. pneumophila phagosomes inhost cells expressing a GTPase-deficient, constitutively activeRab7 mutant.

Studies of populations of isolated phagosomes containinglatex beads (16, 17, 30) have suggested a sequential acquisitionand loss of Rab5 and subsequent acquisition and loss of Rab7.However, at early time points, the populations of phagosomescontaining the inert particles exhibit both markers (17, 30).Because these studies examined populations of phagosomes(rather than individual phagosomes), it is unclear from themwhether an individual phagosome can have both Rab5 andRab7 simultaneously or whether acquisition of Rab5 is a pre-requisite for acquisition of Rab7 by a phagosome. In priorwork, we have found that M. tuberculosis phagosomes exhibit apersistence of Rab5 and that L. pneumophila phagosomesnever acquire Rab5 (13). Combined with our present findings,

these results suggest that in the case of M. tuberculosis phago-somes, both Rab5 and Rab7 can be present simultaneously onthe phagosome and that in the case of L. pneumophila, acqui-sition of Rab5 is not a prerequisite for acquisition of Rab7.However, confirmation of these hypotheses will require exam-ination of cells that simultaneously express both markers atdetectable levels.

Prior studies (33, 38) employing immunofluorescence exam-ination of cells overexpressing Rab7 have found that Rab7exhibits only a limited colocalization with late endosomal-ly-sosomal markers. For example, whereas CD-M6PR is typicallyrestricted to a perinuclear area, Rab7 has been observed toextend to the cell periphery. In our studies, with the resolutionafforded by electron microscopy, it is clear that many vesicles inthe cell that stain richly for Rab7 completely lack LAMP-1 andCD-M6PR immunogold staining. Thus, while functional stud-ies employing constitutively active and negative forms of Rab7have indicated that Rab7 is important in regulating membranetrafficking between early and late endocytic compartments (18,38), it is also clear that much of Rab7 is found on compart-ments that are neither late endosomes nor lysosomes. Onepossible explanation for these findings is that Rab7 is presenton an intermediate compartment or on a shuttle vesicle com-partment between the classical early and late endosomal com-partments and that this intermediate or shuttle vesicle com-partment is rich in Rab7 but has very little of the classical lateendosomal and lysosomal markers. An alternative explanationis that Rab7 may also function in pathways other than the usualendocytic pathway. For example, it may function to promoteautophagy of other organelles (e.g., ER and mitochondria) orto remodel subcellular organelles by regulating fission of mem-brane vesicles from the organelles and the subsequent fusionof these vesicles with late endosomes. In this model, phago-somes and organelles that do not themselves fuse with lateendosomes and lysosomes may undergo remodeling such thatvesicles derived from them are targeted to late endosomes.Either of these models would account for the limited colocal-ization observed between Rab7 and the classical late endoso-mal marker, CD-M6PR.

Our observations of relatively high levels of Rab7 on L.pneumophila and M. tuberculosis phagosomes could be ex-plained by at least two different hypotheses. First, the bacterialpathogens may interrupt the maturation of their phagosomesat a Rab71-LAMP-12 stage by blocking the action of Rab7.Failure of the L. pneumophila and M. tuberculosis phagosomesto mature beyond the Rab71-LAMP-12 stage could be due toabsence of downstream effectors of Rab7 or due to inactivationor inhibition of the function of downstream effectors of Rab7that promote phagosomal maturation. In the case of M. tuber-culosis, which exhibits a persistence of early endosomal mark-ers, this would be at a stage between early and late endosomes.The exclusion of CD-M6PR from the M. tuberculosis phago-some in this study, as well as that of Xu et al. (47), confirmsthat the block in maturation occurs at a stage prior to inter-action of the phagosome with late endosomes. In the case of L.pneumophila, which never acquires early endosomal markers,this stage might represent an early stage of an as-yet-uniden-tified pathway. Noting that L. pneumophila phagosomes andautophagic vacuoles are both surrounded by ER, Swanson andIsberg (43) have previously speculated that the L. pneumophilaphagosomal pathway might reflect an aberrant autophagicpathway. If so, then L. pneumophila may be interrupting mat-uration of an early stage of autophagosome formation, prior toacquisition of late endosomal or lysosomal markers by theautophagic vacuole. While arrested maturation on an autopha-gosomal pathway could account for the presence of Rab7 and

FIG. 9. Quantitation of Rab7 Q67L and LAMP-1 immunogold staining inHeLa-Rab7 Q67L cells 2 days after coincubation with live M. tuberculosis andlatex beads. Monolayers of HeLa cells expressing Rab7 Q67L were coincubatedwith M. tuberculosis and latex beads for 2 h, were washed extensively, wereincubated for an additional 2 days, were fixed, and were processed for immuno-electron microscopy. Immunogold staining for Rab7 Q67L (A) and LAMP-1 (B)was quantitated on phagosomal membranes, nuclear membranes, and plasmamembranes. (A, left panel) Both the M. tuberculosis phagosomal membranes andthe latex bead phagosomal membranes stained positive for Rab7 Q67L. Nuclearmembranes and plasma membranes had negligible levels of staining for Rab7Q67L. (B, left panel) LAMP-1 was scarce on M. tuberculosis phagosomes butabundant on the latex bead phagosomes in these cells. (A and B, right panels) Asa positive control, staining per unit area of cytoplasm was determined for bothRab7 Q67L and LAMP-1. Data shown represent the means and standard errorsof the means of gold particle counts for each compartment. A total of 59 M.tuberculosis phagosomes and 51 latex bead phagosomes were evaluated.



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FIG. 10. In HeLa cells expressing Rab7 Q67L, Rab7 Q67L colocalizes with LAMP-1 on cytoplasmic vesicles, but LAMP-1 remains absent from L. pneumophila andM. tuberculosis phagosomes. HeLa-Rab7 Q67L cells were fixed 2 h after infection with wild-type (A and B) or avirulent L. pneumophila (C), as described in the legendof Fig. 8, or 2 days after coinfection with live M. tuberculosis and latex beads (D, E, and F), as described in the legend of Fig. 9, and processed for immunoelectronmicroscopy. (A) Rab7 Q67L (15-nm immunogold particles; large arrowheads) colocalized extensively with LAMP-1 (10-nm immunogold particles; small arrowheads)on vesicles that appeared to be autophagosomes or multivesicular bodies. The absence of LPS excludes the possibility that the vesicle shown contains L. pneumophila.(B) L. pneumophila phagosomes often stained richly for Rab7 Q67L (15-nm gold particles; large arrowheads) but showed little or no staining for LAMP-1. LAMP-1(10-nm gold particles; small arrowheads) was present on adjacent cytoplasmic vesicles. L. pneumophila LPS was stained with 5-nm gold particles (small arrows). (C)Avirulent L. pneumophila phagosomes frequently stained positive for Rab7 Q67L (15-nm gold particles; large arrowheads) and consistently stained intensely forLAMP-1 (10-nm gold particles; small arrowheads). L. pneumophila LPS was stained with 5-nm gold particles (small arrows). (D and E) M. tuberculosis phagosomes,like L. pneumophila phagosomes, frequently stained richly for Rab7 Q67L (15-nm gold particles; large arrowheads) yet acquired only low levels of LAMP-1 (10-nmgold particles; small arrowheads). Mycobacterial LAM was stained with 5-nm immunogold particles (small arrows). An autophagosome shown on the right side of panelD stains richly for both Rab7 Q67L and LAMP-1. (F) Latex bead phagosomes in these cells, on the other hand, had low to moderate levels of staining for Rab7 Q67L(15-nm gold particles; large arrowheads), but stained intensely for LAMP-1 (10 nm gold particles; small arrowheads). The latex bead phagosome shown in this panelhas one Rab7 Q67L immunogold particle and approximately 20 LAMP-1 immunogold particles. An adjacent vacuole with multiple internal membranes stains positivefor both Rab7 Q67L and LAMP-1. Magnifications are (A) 341,300; (B) 329,400; (C, D, and E) 332,200; and (F) 332,900.



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recruitment of ER to the L. pneumophila phagosome, severalfeatures of the L. pneumophila phagosome are not adequatelyexplained by this model. For example, the L. pneumophilaphagosome develops intimate interactions with mitochondriaand ribosomes, whereas autophagic vacuoles do not. A secondhypothesis to account for the presence of Rab7 but a paucity ofLAMP-1 on the M. tuberculosis and L. pneumophila phago-somes is that Rab7 may be involved in pathways other than theendocytic and phagocytic pathways, such as membrane remod-eling. Hence, the presence of Rab7 on the L. pneumophila andM. tuberculosis phagosomes could reflect active remodeling ofthe phagosomes by Rab7 shuttle vesicles. That Rab7 expres-sion on the L. pneumophila phagosome diminishes after thephagosome completes the formation of its ribosome-lined rep-licative vacuole, in which the organism resides for the remain-

der of its life cycle in the host cell, is consistent with thishypothesis.

ACKNOWLEDGMENTS

We are grateful to Birgitta Sjostrand and Chalermchai Chaloyphianfor expert technical assistance.

This work was supported by a research grant from the AmericanLung Association and by grants AI-31338 and AI-35275 from theNational Institutes of Health. M.A.H. is the Gordon MacDonaldScholar at the University of California, Los Angeles. During the timethat this work was performed, D.L.C. was supported by a YoungInvestigator Award from the Infectious Diseases Society of America.

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FIG. 11. L. pneumophila and M. tuberculosis phagosomes that stained positive for Rab7 had only low levels of staining for the late-endosomal marker, cation-dependent mannose-6-phosphate receptor. HeLa-Rab7 cells were transiently transfected with pZeo-cd-m6pr and incubated for 2 h with L. pneumophila (A) or for 2days with M. tuberculosis (B) as described in the legends of Fig. 2 and 6, respectively. (A) The L. pneumophila phagosome shown has four Rab7 immunogold particles(15-nm gold particles; large arrowheads) but only one M6PR immunogold particle (10-nm gold particles; small arrowhead). M6PR is abundant on cytoplasmic vesiclesoutside of the L. pneumophila phagosome. (B) M. tuberculosis phagosomes stained positively for Rab7 (15-nm gold particles; large arrowheads) but had relatively fewCD-M6PR immunogold particles (10-nm gold particles; small arrowheads). Although the CD-M6PR staining of the M. tuberculosis phagosomes was statisticallysignificant, it was unimpressive when compared with the levels of Rab7 immunogold staining on the M. tuberculosis phagosomes and the level of M6PR staining on othercytoplasmic vesicles (note the vesicle staining intensely for M6PR between the two M. tuberculosis phagosomes). Magnifications are (A) 333,120 and (B) 347,520.



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